DeCyder 2D 7.0 - GE Healthcare Life Sciences
Contents
1. Result after analysis 1 Biomarkers identified 2 Classifiers created 3 New samples classified Workflow for an overview of data The workflow described here helps you to get an overview of the data There are several other possibilities for analyzing data e g partitioning clustering and discriminant analysis The order in which the analyses are made can also vary To move between the different steps in the workflow use the tabs Setup Calculations Results and Interpretation Setup 1 Open the EDA module and click Create Workspace in the Step 1 area on the Setup tab 2 Select and import a matched BVA workspace It is possible to import several matched BVA workspaces Note No other values than log standardized abundance will be imported 3 Check experimental design in the Step 2 Experimental Design area Select a group and click the Edit Group button to make changes Note It is recommended to use different colors and short names on the experimental groups to facilitate the analyses in EDA 4 Inthe Step 3 area click Manual to create the Base set manually as described below e For Protein Filter select of spot maps where Biological Interpretation of biomarkers Result after analysis Biological information for the biomarkers e g molecular functions and which pathways they are part of is obtained Biological Interpretation of proteins from the Pattern Analysis p122. Exp giaups Spot maps or Exp groups ee bo Settings 5 principal components will be calculated DeCuder 7 0 Getting Started Guide 28 9414 49 AA 3 Select Calculation gt Pattern Analysis and algorithm Hierarchial Clustering a In Patterns to be calculated first select the left radio button for Proteins and click Add to list b Then select the left radio button in Spot Maps or Exp groups and click Add to list Pattern to be calculated Proteins q Spot maps or Exp groups Spal maps Hierarchical Clustering settings Distance measure Euclidean Linkage Average Settings 3 Click Calculate to perform the calculations View results 1 To view the results of the calculations go to the Results window 2 Select the Principal Component Analysis tab to view PCA1 and PCA2 ra gt 2218 The Protein score plot above to the left shows if there are any outliers The outliers can be very strongly differentially expressed proteins or mismatched spots and have to be checked in BVA In the Spot Maps loading plot above to the right spot maps that belong to the same experimental groups shall be grouped together If they are not this indicates that something is wrong with the spot map p13 EDA cont 3 3 To see the results of the Hierarchial clustering select the Pattern Analysis and Hierarchial Cluster Analysis tabs in the Results window fs tle fe ee
3. psd Pee 0 DeCuder 7 0 Getting Started Guide 28 9414 49 AA Interpretation Perform interpretation Add Protein ID Perform interpretation To view details of a candidate select a protein candidate in the protein table and click Select Candidate or click the web links Create queries to obtain information on molecular functions of the protein biological processes which pathways the proteins are part of etc For more information see EDA User Manual chapter 11 or the Online Help REE er imej SE ro ic si sted Mrmr ar of Bo ree gar Eara Eriky imaia tata aos aie ro rap hte T T D eiis Fo Tam rated aa compl D p15 Database handling X To move one or several workspaces use export import of data To secure and or move complete databases use backup and restore Only users with Administrator rights in the Database Administration Tool see step 4 on page 2 can perform the operations described below Export Import of data To save a copy of one or several workspaces to a different place it is recommended to export the specific workspacels and import them into the new place Export and Import is performed in the Database Administration Tool All types of workspaces DIA BVA and EDA and gels can be exported and imported The data is exported to the Export folder and is imported from the same folder See MD DeCyder User Manual chapter 10 or the Online Help for additional informa
4. Result after analysis Biological information for the proteins e g molecular functions and which pathways they are part of is obtained protein is present gt 70 Too many missing values protein spots would cause abortion of calculation Base set before filtering Biris ined Diti ick Piar mirar ghrp DeCuder 7 0 Getting Started Guide 28 9414 49 AA EDA cont 2 e Click Add e For Spot map Filter select Remove unassigned spot maps and click Add e Click Apply Filter e Click Create Base Set Base set after filtering Sot To Ee Creal Bente m art LE Trot rape oy mi LIIF atr atenh riaa iie fer 5 Save the EDA workspace via File gt Save workspace Calculations and Results For more details see chapters 7 10 in EDA User Manual or the Online Help Principal Component Analysis and Hierarchical Clustering Perform PCA analysis to find possible outliers and hierarchical clustering to confirm outliers on the base set 1 Inthe Calculations window select the base set in Selected Set 2 Select Principal Component Analysis a In Type of Calculation select Proteins vs Spot maps and click Add to list The calculation will be given the name PCA1 by default b Select Spot maps vs Proteins and click Add to list Type of Calculation Praleins Soal macs Praleins Proteins Q Oe i a Soal maos Pratleins Exo aigues Pialeins Principal Components Analysis settings
5. application User Administration Tool Access groups for User Administration Tool User Administrator TIP Copy the password normally including several special characters before exiting p2 Note By default the password expires after 90 days the account is locked after 3 login attempts with incorrect password and can only be opened again by a User Administrator 6 Finish by clicking OK and close the dialog TIP Add an extra user with unlimited access and without password expiry TIP The DEFAULT_ADMIN user can be used to restore lost users or passwords at a later stage Select Password profile Profile 3 Password never expires Unlimitted login attempts Log in to DeCyder 1 Double click the DeCyder 2D icon y on the desktop or select Start gt All Programs gt GE Healthcare gt DeCyder 2D Software gt DeCyder 2 Log in to the DeCyder application using the user created above and the automatically generated password 3 Atthe first login you will be informed more gt gt Login ouie that the temporary password has expired Click OK to continue and change the password you will be notified on correct password formatting if using wrong formatting DeCyder login Username Password Database DECYDER DeCyder 7 0 Getting Started Guide 28 9414 49 AA Software overview DIA BVA EDA Organizer Differential In Gel Biological Variation Analysis Extended Data A
6. click Finish to complete the Restore backup If there are users logged on the Restore backup logged users window will be displayed Ensure that the users are logged out before you click OK to continue the restore 7 After Restore backup has been performed the application must be closed Click OK in the dialog Note To use the new version 7 0 colors for a restored database change the color settings in DIA and BVA In both modules choose View gt Properties and select Colors gt Default and press OK DeCuder 7 0 Getting Started Guide 28 9414 49 AA Ordering information Getting more help Product Code Number DeCyder 2D 7 0 User Manuals 28 9435 85 Ettan DIGE System User Manual 18 1173 17 DeCyder 2D Software version 7 0 including Extended Data Analysis module Getting Started Guide Installation Guide 28 9435 83 Pre installed computer 28 9435 86 All documents can be viewed at www gelifesciences com DeCuder 7 0 Getting Started Guide 28 9414 49 AA More detailed information can be found in the documentation listed below All user manuals can be found as odf s via Start gt All Programs gt GE Healthcare gt DeCyder 2D 7 0 Software gt Documents and Help unless otherwise stated To find your topic in the pdf use the search function Online helps are accessed via the Help menu via the help buttons in the software dialogs or by pressing F1 DeCyder 2D Software jj Installati
7. modules to perform all stages of the 2 D DIGE analysis process Once the Batch Processor has been set up the gels are processed after each other without user intervention The gels are always warped in the Batch Processor Workflow The normal batch processing workflow contains these steps Set up the DIA batch list 1 Open the Batch Processor module 2 Right click in the workspace opened and select Add DIA item or select File gt Add DIA Batch item 3 Select project and the images to add and use Add or Add all to import images 4 Enter the number of estimated spots to be detected normally 10 000 5 Check Volume in Spot Exclusion Filter area and enter 30 000 for volume 6 Click OK EDA Extended Data Analysis Set up the BVA batch list 7 Setup experimental groups as described above 8 Define spot map function analysis master pick gel and select the best quality standard image as master and press OK The DIA and BVA batch lists are now populated Note Protein statistics and filter can be included in the batch run but it is recommended to run the batch first and then check matching and perform protein statistics and filtering in BVA Save the Batch 9 Choose File gt Save batch Run the Batch 10 Choose Process gt Run batch EDA is used for multivariate analysis of protein expression data derived from the BVA module or the Batch Processor As an addition to univariate analysis you c
8. 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Getting Started Guide 28 9414 49 AA 10 2008 Etteplan Industry AB
9. Bd Gel 5 Treated 2 Hom CyXe Eaj Gel 7 Control 1 Ac CySlecity E4Gel 8 Treated 3 Ac Cy Nedted AF Td Gol 7 Treated 2 CU Cy Bled Ziel 9 Treated 3 Hom CySlecited tf S J Gel Treated 2 Hom CyS Tj Gal 9 Control 2 ac Cy ede 3 5 Treated TIP Assign colors to the different groups e g light blue for control and red for treated by clicking the colored dot and selecting color 5 Define master for matching Check the standard images for quality and select the most representative image average quality not the best not the worst and assign it as master M Optional Landmark gels For normal gel qualities no landmarks are needed Match directly without setting landmarks General recommendations for landmark settings e within or close to distorted region e if possible select spots that are isolated are well shaped circular have medium abundance For more information on landmarking see y DeCyder User Manual chapter 5 or the Online Help E coer Match gels 1 To match the images choose Process gt Match and select Match unmatched and landmarked in the Match dialog To improve matching optimization using warping is included by default in the matching After matching the number of matches is displayed in the Spot Map Table p8 Check and edit matches Check matching 1 Click the M button to switch to Match mode When clicking a spot it is displayed in the 3D Vi
10. EST out files 1 2 3 4 Select File gt Import Protein Information Click Get Pick List and locate the pick list Select search engine and click Add Protein Info Select MS data files click Import Protein Info and Close Note MS files are result files with protein identity information from mass spectrometry and data base searches L Takt EVE Farke E 1 bra Search Caged a imm E b boa Daco ina eerie 4 S menwa pice parser rej sng ane reser wee imt ee Eia sie Dawe d e bh Prime ad tEn bew mw sr i A Fre ia EESE or a bt aL ae er Leo ia aL ab TEUR Fih toi oy ak Wa tia HE oil HLM a Geran te Popeth riat geome poocorted ber AA A Bs ib git aig Panjata brii Fpi aiiai i ktit LIE amg 4 FMHLH Pin ea E EF Er hr ay DVR TOS iih jr ai odie les i amble degian fap awil at ae an Fa a TUNA a ai ah hg meji i M mae i rm us t ul a Lt LEE FEF LLLI PHL ir eres Fpp mariin eie ar Eee yah g Wi Fae de PRR A Mai Rita al fee TA 17g Ear FHL Ju BIRAI AI Mai ruma ar EEDS re a Fan Tif aie fetiprigereds anette Seid mee egani Ha aa Pe re WK ia een FUEL Pe MMR BD Pee ree ay Pry ee i 5 ie Piai Eea riiin HA Pea ere cea Phir pm fie Pen eae Bait pai arte 31s Bid epee Peal ail The SHEE 13 LLH PL Le Eexvar i repos Pie deer Of greri Bop em a5 EPL Ee t The Maara nat ba siid ta mene aiy Prods Die een Dace ipa Fi braiei reumat es ren Bn Pi gent erat a carb i ear Evie e
11. GE Healthcare DeCyder 2D 7 0 Getting Started Guide DeCyder 2D Contents Software overview Recommended workflow Introduction Software preparations Image Loader DIA Differential In gel Analysis BVA Biological Variation Analysis Batch Processor EDA Extended Data Analysis Database handling Ordering information Getting more help N AO oO F amp F fF W N 11 16 17 17 Introduction DeCyder 2D Software is an automated image analysis software suite which enables detection quantification matching and analysis of Ettan DIGE system gels The two dimensional 2D electrophoresis gels are used for separating complex protein mixtures labelled with up to three CyDye DIGE Fluor dyes The multiplex labeling enables detection of small differences in protein levels as well as inclusion of an internal standard The standard is a pool of all the samples within the experiment and therefore contains all proteins included in the experiment By using an internal standard gel to gel variation can be eliminated quantification is accurate and system variation can be separated from biological variation For details on experimental design see Ettan DIGE System User Manual chapter 3 or DeCyder User Manual Software preparations and or EDA User Manual both chapter 1 or the Online Help The Batch Processor enables fully automated processing of multiple gels in the DIA and BVA modules without user in
12. Pick references the warping has to be turned off by clicking the View warp icon Wi 6 In Protein Mode pick positions are Tad displayed in the 2D and 3D images and can be edited Edit gt Edit pick locations For more information on editing pick locations see DeCyder User Manual chapter 5 or the Online Help 3 ei e 2 RE _ we a Ci i z a be b p10 Export pick list to spot picker Export the pick list 1 Select File gt Export Pick list 2 Select pick list and pick spot map and click OK 3 Select location for the pick list file select to save as type txt and click Save Evaluate and export results 1 InP mode select the significantly differentially expressed proteins one by one and check that the matching is correct and that the proteins are relevant In the graph view data points associated with individual proteins are displayed The example below shows results when conditions are used e g a time series analyzed with 2 way ANOVA 2 Export the results Examples of results that can be exported e Tables can be copied to other applications e g Excel e Images mosaic views graphs 3D views can be copied as high resolution images DeCuder 7 0 Getting Started Guide 28 9414 49 AA Batch Processor f When analyzing multiple gels the Batch Processor can be used to automate the process The Batch Processor links both the DeCuder 2D Software DIA and BVA
13. agent impurities dirty glass plates which might affect spot detection and matching Especially images of preparative gels can be very dark and may have to be adjusted Detect spots Process gel images The DIA module processes a set of up to 3 gel images from a single gel Each image is generated from samples labeled with different dyes The DIA algorithm detects spots on a combined image derived from merging individual images from an in gel set of images This co detection ensures that all spots are represented in all images DIA quantitates spot protein abundance for each image and expresses these values as a ratio thereby indicating changes in expression levels by direct comparison of corresponding spots p6 Spot detection 1 Choose Process gt Process gel images and DeCyder spot detection algorithm 6 0 2 For estimated number of spots select 10 000 overestimation to compensate for the detection of non protein objects on the image e g dust particles which are subsequently excluded from the analysis Click OK The spots are now detected and the result is displayed a Sige ee 1 r we yer gt eS ey oes ek Poa hs 2 sade gt Pokaan a8 ey e ae fet Sewer feet ee eee are UERGER UI A EPLE ELI pees SSE ESTE EEE 3 li S Exclude or filter spots Exclude filter 4 Choose Process gt Exclude Filter and select 30 000 for volume recommended exclusion filter for normal qua
14. an perform PCA analysis pattern analysis discriminant analysis and biological interpretations of the results Use of sets in EDA All analyses in EDA are performed on sets data displaying spot maps on one axis and proteins on the other axis Spot maps gt Proteins Set The first step after creating the EDA workspace is to remove standard information not needed in the EDA analyses thereby creating a base set The base set is then further analyzed with different calculation methods resulting in new sets of data These sets can be further calculated and or combined into new sets of data DeCuder 7 0 Getting Started Guide 28 9414 49 AA EDA WS containing the original data set Filtering and normalization rts Base set 1 Calculations Interpretation 2 Selection or filtering of results and or o Combining sets p11 EDA cont 1 Analyses in EDA P He BVA WS EDA WS l Result after analysis Significantly differentially expressed proteins extracted os Differential Expression Analysis Sey PCA Analysis l Data extracted from the analyses a m a AND OR PP Pattern Analysis Result after analysis Proteins with similar expression profiles identified J Data set containing Data set containing biomarkers relevant data i w T l Result after analysis Dato overview Outliers in the data set identified o fs Discriminant Analysis
15. ck Create Further calculations on the new set Perform multivariate analysis A set with significantly differentially expressed proteins has been created Principal Component Analysis and Hierarchical Clustering can be performed on the new set In the calculations window Select set select the newly created set and then the type of calculations to be made Example Hierarchical clustering of the set created after filtering of T test aes a yd T E Fe Ei There are several other calculations that can be made The following example shows an experiment including time series analyzed with partition clustering Each cluster contains proteins with the same expression profile Sets can be created for one or several of these groups for spot picking odie re eo acra hater sS amro Gime y ye pa re tt F FEF DeCyder 7 0 Getting Started Guide 28 9414 49 AA EDA cont 4 Create pick list 1 Select a set containing the interesting points that Import protein information you want to identify and click Filter set The set can be created e g based on the result of the statistical analysis Select Tools gt Create pick list in BVA enter a name and click OK BVA opens with the proteins in the set assigned as pick Export the pick list as described in the BVA section see page 10 Export pick list to spot picker Protein information can be imported from e g Mascot dat files and SEQU
16. ct next image and the crop tool The most recently used cropping rectangle is shown and can be adjusted 8 Save one edited image at a time or save all using Save all The edited images are placed in the same directory as the original images with edited added to the name 9 When the images have been imported exit the Image Editor 10 Check settings Dye Chemistry etc and change if required All red texts must be edited before import Note The settings can only be changed before importing the image 11 Click Import 12 Exit from the Image Loader Note When cropping gels for picking the pick gel must still contain the reference marker To facilitate proper matching define an area of interest in the DIA module instead See DeCyder User Manual chapter 4 or the Online Help p5 DIA Differential In gel Analysis The DIA Differential In Gel Analysis is the initial step in DeCuder analysis and performs e spot co detection e spot quantification by normalization and ratio calculation The DIA module removes most of the system variations Create DIA ws Create a DIA workspace A DIA workspace contains information from one gel 1 Open the DIA module and select File gt Create Workspace 2 Select project all images of one gel and click Create Viewing contrast brightness settings Adjust the contrast brightness settings to see if there are any image disturbances such as Newton rings re
17. d into a Gel named GelO1 Gel 01 Standard Cy2 gel e Gel 01 Time1_Dose2 Cy3 gel e Gel 01 Time2_Dose2 Cy5 gel If gel images are not named as recommended they will not be automatically sorted See y DeCyder User Manual chapter 3 or the online help for information on how to manually group gel images TIP Use short names with the most important part of the name first Loading and editing images 1 Open the Image Loader module 2 Select an existing project to load the images to or create a new project Click Add and browse for images Select all images to be loaded and click Open Select the images to be edited and click Edit Gel Images to open the Image Editor Images hi imper d ht geij ada basara inara Ge Gpe cherry Image Gyi Am Une REJ she hee Pe Iming Lyt 6 Use the Crop tool 4f to define a crop area on the first image Adjust the area as required and double click inside the crop area to crop Me 68 View Toas Widows Help Baer Galt so HAF GE Ceni 75917 DeCuder 7 0 Getting Started Guide 28 9414 49 AA Cropping recommendations e Reduce the image as much as possible e All areas that do not contain usable information should be removed Ensure that all relevant spots remain inside the image e Itis more important to ensure consistent patterns than equal sizes e Similar patterns facilitate correct matching TIP Images can be rotated using the tools Flip and Rotate 7 Sele
18. e or in the Protein Table to display it in the 3D View Note To be able to see a spot in 3D View the spot map has to be selected and displayed in the Image View To select the first image left click on the spot map header To select the second image right click on the spot map header DeCuder 7 0 Getting Started Guide 28 9414 49 AA Se as tss m an j eapi CP ae g ELL at i e a Typical example of a differentially expressed protein when comparing the control group with the treated group Use Protein filter to assign protein of interest Assign protein of interest using Protein Filter 1 2 cy In P mode choose Process gt Protein filter Selection see Protein Filter dialog below Click the Filter button The number of filtered proteins is displayed If necessary change settings and click Filter again When ready click OK Choose View gt Properties select Protein Table gt Protein Table Filter gt Protein of Interest to show only filtered proteins assigned as protein of interest Example 1 Recommended filter settings to filter for differentially expressed proteins between two selected experimental groups In this example the filter is restricted to proteins which are present in at least 9 out of 12 spot maps 3 of 4 gels Fie aon eA ol irme M As Pek n in ki LT Lai f Densil fiba imttirgye M Sasal Aes m Genin piim fe feka in poles predentin E lect
19. ew it is highlighted in the Table View and all spots that are matched to the spot are marked with magenta spot boundaries in the images The images can be viewed unwarped or warped If warping has been performed this is displayed by default Right click in an image and select to show or hide warp grid spot contours match vectors and or colored overlays See also A DeCyder User manual chapter 5 or the Online Help A warped image with warp grid and overlay J S ia N y a displayed is shown to A SAR aes the right A i y 2 Use zoom and scroll functions to adjust the image view e 2D image matched are green autolevel 1 or lilac autolevel 2 unmatched orange e 3D graph master spot map and selected spot map To select a spot map click the spot map header or on a spot in the spot map e Match table autolevel 1 and 2 unmatched see DeCyder User manual chapter 5 or the Online Help 3 Remove matches in areas with incorrect matches e g areas where the match vector directions differ from the overall vector direction by selecting the match spot and clicking Break Match 4 Select a number of spots to check the matching performance Select the spot one by one on the Master gel and compare the matching on the other gels DeCuder 7 0 Getting Started Guide 28 9414 49 AA BVA cont 2 If required correct individual matches using the editing tools at the bottom of the BVA window Contin
20. in Saian piian wath Fe Sander Tii valia wn FF design Fee fis F orisa Fato am F TE me M Ones AHD volo SaN wele r i T E aa rs rF E kas Properties lor protein in pick spot map Sr gj C pl CE F ra aa EN rer nto 7258 pm OF Cancel Help p9 BVA cont 3 5 Select the significantly differentially expressed proteins one by one in the Protein Table and check that the matching is correct and that the proteins are relevant If required correct individual matches in M mode see page 8 Check Matching Note Both Protein of Interest and Pick can be set manually using the checkboxes POI and Pick in P mode Create a pick list 1 If not already in the workspace import a DIA workspace from a preparative gel using File gt Add Template DIA Workspace 2 In Spot mode set function Pick P on this gel by selecting the image and checking Pick P 3 Match this gel POls from the master gel are transferred to the pick gel Landmarks can be needed to match the pick gel 4 Assign all proteins of interest as pick by selecting Process gt Assign All Protein of Interest as Pick or set pick manually in P mode by selecting the spot in the pick gel and check Pick 5 If no picking references have been defined in DIA define the references by choosing Edit gt Define Picking Reference and define both Picking references See DeCyder User manual chapter 4 or the Online Help Note To be able to define and or edit
21. ion Tool used for setting up users and their rights See page 2 ER Database Administration Tool used for backing up the database etc See page 14 DeCyder 7 0 Getting Started Guide 28 9414 49 AA p3 Recommended workflow Load and crop images Create DIA ws Detect spots lt gt Exclude or filter spots Create BVA ws from DIA ws Set up exp design Match with without landmarks Check and edit matches Create EDA ws from BVA ws Perform statistics and filter proteins Perform statistics and filter proteins Perform multivariate analysis Assign proteins for picking Assign proteins for picking Export pick list to spot picker Protein identification Add Protein ID Add Protein ID Evaluate results Evaluate results O Image Loader DIA Batch automation of BVA DIA and BVA Outside DeCyder ws wo rkspace Perform interpretation p4 DeCyder 7 0 Getting Started Guide 28 9414 49 AA Image Loader Image loading DeCuder software uses an Oracle Database Therefore the images must first be uploaded to the database before they can be analyzed Gel name recommendations Image Loader automatically groups up to three images into one gel if the images are named with a gel number e g Gel 01 a description of the content e g dose 1 or time 3 in parentheses and the dye used e g Cy3 For example these 3 gels will be groupe
22. jaich Sat ede Mitch Hatch Crassus F Hea F Hipi Epid O Spk d keah Capa ta Hazia OELE Hull Soaked ogy te Hatch Amava from Match or enhance the landmarking and then rematch the images For more information on detection and matching see M DeCyder User Manual chapter 5 or the Online Help 5 If many corrections have been made re match the gels choose Process gt Match and select Match unmatched and landmarked Statistical analysis and filter proteins Perform statistical analysis on analytical gels included in the experimental design The gels must have been sorted into experimental groups see page 7 1 Click the P button to switch to Protein mode 2 Choose Process gt Protein Statistics and check Average ratio and Student s t test for the experimental groups to compare e g Control vs Treated Select groups in Population 1 and 2 Type of awn trees E rdependert torts normal Buted touts fuse tamci Gao bo gap orpoa Average Bato T hidis Tier Papiston 1 Opora voted 4 Member Mape gap Companions M Onewa ANOVA Deteeen ditherent gops M mowa ANOVA beween Condoni and Condhong T Apoi tate decover ute FDR comecten Rate trots ae adobe 3 If more than two experimental groups are analyzed select additionally One Way ANOVA 4 Click Calculate The display mode will switch to P and the results of the Statistics are now displayed in the Protein Table Select a spot in an imag
23. l e The spot maps in each experimental group have been clustered together as displayed in the dendrogram above the image and with the color marked spot maps below the image e Arough estimation of the number of protein groups with the same expression patterns can be made from the dendrogram to the left of the image and filter proteins The next step is to analyze the base set to find differentially expressed proteins Differential Expression Analysis 1 Inthe Calculations window select the calculation Differential Expression Analysis 2 Select type of statistical test e g Student s T test 3 Click Add to List and then Calculate to start the calculation As the data set contains a high number of non differentially expressed proteins noise the results of the Student s T test will be used to filter out the differentially expressed ones new set creation Create new set for further calculations Based on the results from the Differential Expression Analysis create a new set for further calculations 1 Click Filter Set 2 Select Protein Filter Student s T test lt 0 05 to extract all proteins with a p value lt 0 05 Protein Filter Select filter criteria Value Student s T test vi lt o 05 EE a Filter Criteria Student s T test lt 0 05 Combine filters C Ce fs CO p14 3 Click Add and then Apply Filter 4 Click Create Set enter a name and color for the new set and cli
24. l Electrophoresis 2 D DIGE technology is covered by US patent numbers US6 043 025 US6 048 982 US6 127 134 and US6 426 190 and foreign equivalents and exclusively licensed from Carnegie Mellon University CyDye This product or portions thereof is manufactured under licence from Carnegie Mellon University under US patent number US5 268 486 and other patents pending The purchase of CyDye fluors includes a limited license to use the CyDuye fluors for internal research and development but not for any commercial purposes A license to use the CyDye fluors for commercial purposes is subject to a separate license agreement with GE Healthcare GE Healthcare has patent applications pending relating to its DeCuder software technology European patent application number EP1 234 280 2008 General Electric Company All rights reserved First published Oct 2008 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences KK Sanken Bldg 3
25. lity gels can be adjusted for different quality and click OK Do not apply the other filters For normal quality images other filters are not needed Save the workspace 5 Choose File gt Save workspace 6 Select a project to store it in or create a new project 7 Type a workspace name and click Save After exclusion of non protein objects the DIA module is completed This analysis can be repeated for all gels that shall be included in the evaluation but we recommend using the Batch Processor module for multiple gels see page 11 DeCuder 7 0 Getting Started Guide 28 9414 49 AA BVA Biological Variation Analysis The Biological Variation Analysis BVA e processes multiple gel images e performs gel to gel matching of spots allowing quantitative comparisons of protein expression across multiple gels There are 4 modes in BVA with different functions Mode Function Rows in table represent S s Set up experi Spot maps corresponding to Spot map mental design gel images M M Inter gel Matched unmatched spots Match matching e Examine statistics Proteins spots in Master Spot Protein of all proteins Map A A Examine statistics Spot maps all spot maps Appearance of one protein where the protein is present See also DeCyuder User Manual chapter 5 Create BVA workspace and import DIA workspaces 1 Open the BVA module and choose File gt Create Workspace 2 Selec
26. mpany Cy CyDye DeCuder and Ettan are trademarks of GE Healthcare companies DeCyder includes an Oracle 10g database Copyright 1995 2007 Oracle Corporation All rights reserved Third Party Credit statements Oracle is a registered trademark of Oracle Corporation and or its affiliates This product includes the Xerces XML parser Copyright 2000 The Apache Software Foundation http www apache org All rights reserved This product includes parts developed using ITK Copyright 1999 2003 Insight Software Consortium for licensing information see http www itk org Parts of this product implement the libTiff library Copyright 1988 1997 Sam Leffler Copyright 1991 1997 Silicon Graphics Inc http wwwlibtiff org Parts of this product include NMath components from CenterSpace Software LLC Copyright 2002 2008 All rights reserved http www centerspace net Parts of this product include components from Farpoint Technologies Inc Copyright 1991 2007 All rights reserved http www fpoint com Parts of this product include the TeeChart component from Steema Software SL Copyright 1995 2008 All rights reserved http www teechart com Parts of this product include the open source MD5 implementation by L Deutsch Copyright 1999 2000 2002 Aladdin Enterprises All rights reserved Parts of this product include Cool Scrollbar Library Copyright 2001 J Brown CyDye 2 D Fluorescence Difference Ge
27. nalysis For File manager for Analysis Co detect Match multiple images from multivariate analysis of protein DeCyder database and quantitate protein different gels to provide expression data derived from BVA Move copy delete spots on a set of statistical data univariate Apart from univariate analysis rename files images from the same analysis on differential protein you can perform PCA analysis projects etc gel from a 2D DIGE expression levels between pattern analysis discriminant See DeCyder experiment experimental groups analysis and biological User Manual See page 6 See page 7 interpretations of the results chapter 2 or the See page 11 Online Help DeCyder r DeCyder 2D Organizer E Help Ylofdyfe Variation Analysis Diffare lal yal Angy Extended D ta Analysis Batch Processor KXMLUTOOIDOX XML Toolbox Image Loader Extract user specific data to Load gel images into the Batch Processor facilitate automatic report DeCyder database to make Automation of the DIA and BVA generation them available for the other module Recommended if you modules have many gels Detect spots match multiple gels perform statistics and filter proteins without user interaction Online Help Software help provided in all modules Click the help button or F1 when you need more information See page 5 See page 11 Other tools TIP Click the icon to open the module User Administrat
28. on Guide Information on how to install the software User Manual Basic methodology for additional information see M Ettan DIGE System User Manual Extensive information on all modules exclusive EDA included in the software Tutorials for more extensive training and understanding Online Help Detailed information on all modules windows dialogs and menus via the context sensitive help function included in the software Access the Online Help via the help menu via the help buttons in the software dialogs or press F1 DeCyder Extended Data Analysis module User Manual Extensive information on the usage of the software and on the possibilities in EDA Tutorials for more extensive training and understanding Online Help _ Detailed information on the usage of the software via the built in context sensitive help function included in the software Access the Online Help via the help menu via the help buttons in the software dialogs or press F1 Ettan DIGE System User Manual Extensive information on two dimensional difference gel electrophoresis View the document at www gelifesciences com p17 For contact information for your local office please visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com GE imagination at work GE imagination at work and GE monogram are trademarks of General Electric Co
29. t a project and DIA then select DIA workspaces you want to include and click Add 3 Click Create to include the images and spot maps from the selected DIA workspaces in the BVA workspace 4 Choose File gt Save workspace 5 Select a project to store it in or create a new project 6 Type a workspace name and click Save Viewing and sorting spot maps The spot maps included in a workspace can be viewed and sorted in various ways DeCuder 7 0 Getting Started Guide 28 9414 49 AA Default sorting New workspaces display all standard spot maps in S and M mode by default When moving to P mode the spot maps are by default sorted after experimental groups When returning to M mode the sorting from P mode is kept A floating master window i e it can be moved separately is displayed in M mode regardless of sorting Custom sorting To select different sorting use View gt Browse Sort Gel Images or click the icon 1 Customized sorting is kept when moving between modes For more details see M2 DeCuder User Manual chapter 5 or the Online Help Number of images to view Select View gt Display Multiple Gel Views or click the icon and select number of images to view or use the gel mosaic horizontal vertical zoom buttons o pa x7 Scrolling in multiple views Use the scroll tool to move among the images ago wj Click in the middle of the scroll tool to open the Image Layout dialog showing in blue
30. tervention The software also includes DeCyder Extended Data Analysis Software used for univariate and multi variate analysis of large and combined data sets Information from protein databases can be linked into the software Getting started This Getting Started guides you through a typical workflow in DeCyder 2D Software You can use either your own gel images or the tutorial gel images in the workflow Installation See DeCyder 7 0 Installation Guide for instructions on how to install the software Create a new user 1 Log into User Administration Tool via Start gt All Programs gt GE Healthcare gt DeCyder 2D Software gt User Administration Tool or by clicking the icon 4 When logging in for the first time use the default user User name DEFAULT_ ADMIN Password DECYDER_00 2 Click New enter login name full name and click OK 3 Click Generate password to get a temporary password Select application DeCyder and check the appropriate Access groups Select application e Scientist access to the DeCuder application only e Administrator access to the Database Administrator administration tool only Scientist And Administrator oO Scientist e Scientist and administrator access to both the DeCuder application and the Database administration tool 5 For users that must be able to add and remove users etc select application User Administration Tool and check User Administrator Select
31. tion Backup To secure the data in the DeCyder 2D database perform a backup regularly 1 Open the Database Administration Tool via Start gt All Programs gt GE Healthcare gt DeCyder 2D Software gt DeCyder Database Admin Tool or click the icon fig and log in 2 Click Manual backup and check that no users are logged in 3 Click Next gt gt and then Finish to start the backup The backup will take some time Backup files are placed in the Export folder 4 IMPORTANT Move the dmp file from the Export folder to a safe place after backup i e not at the same computer as the database and preferably a storage with regular backup p16 Restore from backup Restore the DeCyder 2D database to exchange the information in the database with an earlier backup It is recommended to perform a backup of the database before restoring the earlier backup Caution Existing data will be overwritten when restoring a backup Note Users that are logged on during the restore will lose their data and will be logged out during the process 1 Ensure that the backup file is placed in the Export folder 2 Open the Database administration Tool and log in 3 Click Restore backup in the Database administration Tool 4 Ifyou have not performed a backup click Yes otherwise click No 5 Browse to the Export folder and select the backup file to restore from and click Open 6 Check that the correct file is selected and
32. which area of the image view is currently displayed Image Layout X Assign experimental groups For more details on experimental design see Ettan DIGE System User Manual chapter 3 or DeCyder User Manual chapter 5 or the Online Help 1 Inthe BVA window click the S button to switch to Spot Map mode 2 Inthe Experimental Design View add experimental groups e g Control and Treated Click Add enter group name and click Confirm Note Conditions e g time and dose can be defined to allow for a 2 way ANOVA analysis See DeCyder User Manual chapter 5 or the Online Help 3 Click on the Unassigned folder to display the list of images to the right p7 BVA cont 1 4 Drag and drop images in the column to the right to the appropriate group folders All standards are automatically included in the Standard folder if named standard std or pooled in the file name Experimental Design View Gy Standeed Unassigned Gad Gel 1 Control 1 CU CyBedit Get 2 Treated 1 Ac CyMecited AF Tj Gel 1 Treated 2 Ac CySled Sd Gel 2 Control 2 CU CySledt Sicet 3 treated 1 Hom CySledted cf Tj Gal 2 Treated 1 Ac Cy2 ede Zj Gel 3 Control 3 CL CySlede T jGas Treated 1 OL Cyedted oF Tf Gel 3 Treated 1 Hom Cyto Tj Gel 4 Control 1 Hom CyMec SiGel S Treated 2 Hom CyNedted tF Zad Gel 4 Treated 1 CU CySied Ed Gel 5 Control 3 Ac CySledi EiGel 7 Treated 2 OU CySedted tF
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